1Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.

Abstract

The retrograde movement of tRNAs from the cytoplasm to the nucleus occurs constitutively in eukaryotic cells but its functional significance remains unclear. We show evidence suggesting that in Saccharomyces cerevisiae, a spliced tRNA precursor must be imported into the nucleus before the biogenesis of a modified base can occur. Wybutosine (yW) is a modified base adjacent to the anticodon of tRNA(Phe) and is required for accurate decoding. Glucose starvation or overexpression of the nuclear tRNA binding protein Trz1p both caused nuclear retention of cytoplasmic tRNAs, impaired the yW synthesis, and induced the accumulation of its intermediate, N(1)-methylgunanosine (m(1)G), showing that the postspliced tRNA(Phe) is imported to the nucleus, where m(1)G is formed by Trm5p, after which it is reexported to the cytoplasm, where the yW synthesis is completed by cytoplasmic enzymes.

Secondary structures of precursor and mature tRNAPhe from S. cerevisiae and the biosynthesis of wybutosine (yW). (A) Secondary structures of the precursor (Left) and mature (Right) forms of S.cerevisiae cytoplasmic tRNAPhe. The modifications are shown. With regard to the precursor tRNAPhe, it is the intron-bearing precursor that accumulates in the mutant rna1 (2). The unmodified positions G10, C32, G34, and G37 are boxed, arrows indicate the splicing sites, and the intron sequence is shown in small letters. In the mature tRNAPhe, the yW modification is boxed. (B) Pathway of yW biosynthesis in S.cerevisiae tRNAPhe. The chemical structures of guanine (G), N1-methylguanine (m1G), and wybutosine (yW) are shown along with the modification enzymes and the locations in which the modifications occur. Thus, Trm5p methylates G37 in the nucleus to generate m1G. In the cytoplasm, yW37 is synthesized by sequential reactions catalyzed by Tyw1p, Tyw2p, Tyw3p, and Tyw4p.

Glucose starvation induces the accumulation of m1G37-bearing tRNAPhe in the nucleus. (A) LC/MS analyses of the total nucleosides in the tRNAPhe molecules isolated from yeast cells (BY4742) after 0h (uppermost panels), 1h (second panels), 2h (third panels), and 3h (fourth panels) of glucose starvation. As a control, the nucleosides of the tRNAPhe molecules that were isolated from ΔTYW1 are shown in the lowermost panels. The Left panels show the mass chromatograms of the base-related ion () of monomethylated guanosine (m/z 166), which detect m1G and m2G. The Right panels show the mass chromatogram of the proton adduct of yWpA (m/z 838). (B) LC/MS RNA fragment analyses of the RNaseA-digested tRNAPhe molecules. The panels show the mass chromatograms that detect the doubly charged ions of the anticodon-containing fragments that bear m1G (Left panels, m/z 1,013.65) and yW (Right panels, m/z 1,119.20). The vertical axes display the apparent ratio of m1G or yW, which is calculated on the basis of the intensity of the m1G- and yW-containing fragments. (C) The TRM5-GFP (Upper) and TRM5-NES-GFP (Lower) constructs are shown. The photos show the phase contrast (PH), GFP-fluorescence, Hoechst 33342, and merged images for each strain. (D) LC/MS analyses of the total nucleosides in the tRNAPhe molecules that were isolated from the TRM5-GFP (Upper) and TRM5-NES-GFP (Lower) strains after 4h of glucose starvation. The Left and Right panels show the mass chromatograms of the base-related ion () of monomethylated guanosine (m/z 166) and the proton adducts of yWpA (m/z 838), respectively.

Accumulation of nuclear m1G37-bearing tRNAPhe after the overexpression of Trz1p. (A) The tagged Trz1p (indicated by the arrow) that was obtained after affinity purification by using IgG sepharose was analyzed by SDS-PAGE. Shown are the whole cell extract (lane 1), the flow through (lane 2), and the eluted IgG sepharose fraction (lane 3). The heavy chain of IgG is asterisked. (B) The RNA components extracted from the tagged Trz1p fraction (lane 2) were separated on denatured PAGE. The pre-tRNAs assigned are indicated. Total RNA (lane 1) is shown for comparison. (C) LC/MS RNA fragment analyses of the RNase A-digested spliced tRNAPhe molecules that were isolated from the RNAs that coprecipitated with the tagged Trz1p. The Left and Right panels show the mass chromatograms that detect the doubly charged ions of the anticodon-containing fragments that bear m1G (Left panel, m/z 1,013.65) and yW (Right panel, m/z 1,119.20), respectively. The vertical axes display the apparent ratios of m1G to yW, which were calculated from the intensities of the m1G- and yW-containing fragments. The asterisk indicates an unassigned RNA fragment. (D) LC/MS analyses of the total nucleosides in the spliced tRNAPhe molecules that were isolated from the Trz1p plasmid-harboring yeast strain after it was grown in conditions that repressed (Upper) or induced (Lower) plasmid expression. The left and righthand panels show the mass chromatograms of the base-related ion () of monomethylated guanosine (m/z 166) and the proton adduct of yWpA (m/z 838), respectively.

Schematic depiction of the tRNAPhe maturation pathway in S. cerevisiae. The primary transcript of tRNAPhe (pri-tRNAPhe) is end-matured by 5′/3′ trimming and the addition of CCA to form precursor tRNAPhe (pre-tRNAPhe). Ten positions are then modified in the nucleus, after which the pre-tRNAPhe is exported to the cytoplasm, where the intron is removed by the splicing machinery at the mitochondrial outer membrane. The spliced tRNAPhe is then modified by the cytoplasmic methyltransferases Trm11p/Trm112p and Trm7p, which make the m2G10 and Cm32/Gm34 modifications, respectively. The tRNAPhe is imported into the nucleus, where G37 is modified into m1G37 by Trm5p. The postspliced m1G37-bearing tRNAPhe is then reexported to the cytoplasm, where yW37 is generated through consecutive reactions catalyzed by Tyw1p, Tyw2p, Tyw3p, and Tyw4p.